Mise en contexte
Il est bien connu que le ribozyme VS reconnait son substrat par la formation d’une interaction kissing-loop dépendante du magnésium qui implique les boucles terminales I et V localisées respectivement dans le substrat et le domaine catalytique. Les structures des domaines I et V sous leur forme libre ont précédemment été caractérisées, révélant une boucle terminale désordonnée pour le domaine I [111] et une boucle terminale contenant un motif de type U-turn pour le domaine V [113, 114]. Deux structures du domaine V ont été obtenues par RMN : la première en absence (SLVfree, [113]) et la deuxième en présence de magnésium (SLVMg, [114]). Ces deux structures ont
révélé qu’en présence de magnésium, le motif U-turn de la boucle V devient plus compacte qu’en absence de magnésium avec un changement de conformation important du nucléotide U700 [113, 114]. En effet, en absence de magnésium, le U700 est situé dans le sillon majeur, alors qu’en présence de magnésium, celui-ci est positionné au sillon mineur. Le nucléotide U700 se retrouve ainsi sur la face de la boucle V qui interagit avec le substrat. Cette observation nous a mené à poser l’hypothèse suivante : le nucléotide U700 est impliqué dans l’interaction kissing-loop I/V.
Nous avons ainsi réalisé une étude visant à mieux comprendre le rôle de la boucle V dans la reconnaissance du substrat. Étant donné que les nucléotides U696, G697, A698 et C699 ont déjà été associés au motif U-turn, ainsi qu’aux trois paires de bases W-C de l’interaction kissing-loop, nous nous sommes penchés sur les nucléotides dont le rôle n’avait pas été étudié précédemment, notamment celui du U700 et de la paire de bases qui ferme la boucle (U695-A701). Pour se faire, nous avons étudié plusieurs variantes de la boucle de SLV dont certaines contenaient une modification du nucléotide U700. Ce dernier a été remplacé par les trois autres nucléotides, A, G ou C. Nous avons également étudié d’autres variantes de la boucle V contenant, par exemple, une plus grande boucle ou encore une paire de bases différente pour fermer la boucle. Nous avons évalué l’impact de ces différentes variations sur l’activité du ribozyme à l’aide d’expériences de cinétique enzymatique. De plus, ces variantes de SLV ont été
caractérisées par RMN pour vérifier leur impact sur la structure de la boucle V, particulièrement au niveau du motif U-turn. Cette étude a permis de démontrer que remplacer la base du U700 par un A, G ou C n’affecte pas significativement l’activité du ribozyme, alors que sa délétion entraîne une grande diminution de celle-ci. Ceci suggère que le squelette ribose-phosphate du nucléotide 700, et non sa base, est important pour l’interaction kissing-loop. De plus, nous avons utilisé la modélisation par homologie pour obtenir un modèle de l’interaction kissing-loop I/V qui nous a ensuite permis de proposer de nouvelles interactions entre les domaines I et V. Cette étude a également permis de développer un protocole RMN simple, basé sur la détection des déplacements chimiques inhabituels du 31P et du 15N N7, pour identifier des motifs U-turn.
Dans le cadre de cette étude, j’ai réalisé la caractérisation RMN de tous les ARN représentant chacune des variantes de SLV. Ceci inclut premièrement la synthèse in vitro et la purification de tous ces ARN. J’ai ensuite procédé à l’enregistrement de tous les spectres RMN du proton, du phosphore et de l’azote de chaque variante de SLV en absence et en présence de magnésium. J’ai ensuite analysé et attribué chacun des spectres RMN. Les expériences de cinétique enzymatique ont été effectuées par Julie Lacroix-Labonté, étudiante au Ph.D et co-auteure de cette étude. La modélisation a été effectuée par le Dr Pascale Legault, directrice du laboratoire, et Geneviève Desjardins, étudiante à la maitrise, avec l’aide de Véronique Lisi, étudiante au Ph.D dans le laboratoire du Dr François Major. L’analyse des résultats s’est effectuée conjointement avec le Dr Pascale Legault. Pour la rédaction du manuscrit, j’ai rédigé une partie du matériels et méthodes, fait les figures de la partie RMN et le reste a été principalement rédigé par le Dr Legault.
Role of SLV in SLI Substrate Recognition by the
Neurospora VS Ribozyme
Patricia Bouchard,1,4 Julie Lacroix-Labonté,1,4 Geneviève Desjardins,1 Philipe Lampron,1 Véronique Lisi,3 Sébastien Lemieux,2,3 François Major,2,3 and Pascale Legault1
1Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-
Ville, Montréal, QC, Canada, H3C 3J7
2Département d’Informatique et de Recherche Opérationnelle, Université de Montréal,
C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada, H3C 3J7
3Institut de Recherche en Immunologie et en Cancérologie 4These authors contributed equally to this work.
This research was originally published in RNA journal:
ABSTRACT
Substrate recognition by the VS ribozyme involves a magnesium-dependent loop / loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U- turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI / SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual 31P and 15N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI / SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I / loop V interface.
INTRODUCTION
RNA hairpins play many essential roles as basic elements of RNA structure. They guide the folding of RNA, help maintain its structural integrity, and serve as recognition motifs in RNA-RNA and RNA-protein interactions. RNA hairpins have recently gained increasing interest given the central role played by microRNAs and various other non-coding RNAs in the regulation of gene expression [1, 2]. Therefore, it is important to understand how these small RNA building blocks assemble to form complex RNA-based architectures and support important biological functions.
Small ribozymes, such as the hammerhead, hairpin, HDV, and VS ribozymes, represent simple RNA architectures capable of enzymatic activity, and have been widely used as model systems to understand structure-function relationships in RNA. The VS ribozyme is one of these few naturally-occurring ribozymes, which possesses a unique tertiary structure to perform its functions and has the distinct ability to recognize a hairpin substrate through formation of a loop / loop tertiary interaction [for recent reviews, see [3] and [4]]. Its catalytic activities are metal-dependent phosphodiester bond cleavage and ligation reactions, which both involve 5'-OH and 2',3'-cyclic phosphate termini [5, 6]. Although the natural VS ribozyme isolated from the mitochondria of certain strains of Neurospora performs self-cleavage activity [5], VS ribozyme fragments of ~100-160 nucleotides can perform cleavage in trans when incubated with small hairpin substrates in vitro [7].
There is currently no high-resolution structure of the VS ribozyme, but structural information is available from the proposed secondary structure (Fig. 2.1A; [8]), tertiary structure models [9, 10], and NMR structures of individual stem-loops [11-16]. The catalytic domain of the VS ribozyme consists of five stem-loop subdomains (SLII-SLVI; Fig. 2.1A), and stem-loop I (SLI; Fig. 2.1B) defines the substrate domain [8]. The cleavage site is located within the internal loop of stem-loop I, between nucleotides G620 and A621 [5]. Our present understanding is that in order for cleavage to occur, the cleavage site internal loop must dock in a cleft formed by SLII and SLVI to allow its
interaction with the active site formed by the A730 loop of SLVI [3, 4, 17, 18]. The proposed catalytic mechanism for site-specific cleavage relies on the direct participation of G638 from the internal loop of SLI and A756 from the A730 loop of SLVI in general acid-base catalysis [19].
Substrate recognition by the VS ribozyme involves a loop / loop interaction between SLI and SLV, which is stabilized by magnesium ions (Mg2+). Mutational
studies have provided evidence for three Watson-Crick base pairs involving nucleotides 630-632 of loop I and nucleotides 697-699 of loop V [20]. Formation of this tertiary interaction is accompanied by a rearrangement of SLI from an unshifted to a shifted conformation (Fig. 2.1B), which affects the structure of the cleavage site internal loop [21, 22]. SLI mutants that cannot adopt the shifted conformation are not cleaved by the VS ribozyme, whereas those that can adopt the shifted conformation are active in the cleavage reaction (Figs. 2.1B-C; [21]). Three-dimensional structures of small hairpins containing the unshifted (inactive) and shifted (active) internal loop conformations have been determined by NMR spectroscopy [11-13]. It appears from these structures that the active conformation contains a unique Mg2+-binding site and a unique tertiary interaction motif, both of which may be important for catalysis [13].
To better understand the role of the SLV receptor in SLI recognition, we have determined two NMR structures for an SLV fragment (Fig. 2.2), one in the absence (SLVfree; [15]) and one in the presence of magnesium ions (SLVMg; [16]). The loop of SLVfree forms a loose non-canonical U-turn motif, whereas that of SLVMg forms a compact canonical U-turn motif [15, 16]. The U-turn of SLVfree was termed non- canonical because it lacks the stacking interaction between the U696 base and the A698 5'-phosphate and the hydrogen bond between the U696 H3 and the A698 3'-phosphate (Fig. 2.2) that are characteristic of canonical U-turn structures and found in SLVMg [15,
16]. Although Mg2+ ions affect the loop conformation, they do not significantly change
the conformation of the three SLV bases (G697, A698, C699) that are proposed to base pair with SLI [20]. In both structures, these three bases of SLV are exposed and well
ions occurs at the extruded loop residue U700, which comes closer to the three interacting bases (G697, A698, and C699) of SLV (Fig. 2.2; [16]). Given this Mg2+- dependent conformational change of U700 and the fact that Mg2+ ions stabilize the loop I / loop V interaction, we hypothesized that U700 could play an important role in SLI recognition [16].
In the present manuscript, we explored the role of U700 and other SLV features (loop-closing base pair, nucleotide insertion) in SLI recognition. We first performed kinetic experiments with the VS ribozyme containing mutations in loop V to better understand the SLV loop requirements for catalysis. To analyze the effects of these mutations on the structural integrity of the U-turn structure in the SLV loop, we developed an NMR-based assay to allow quick structural mapping of small SLV fragments. Given that the structure of SLVMg is available and that structural characteristics of the SLI loop have been previously derived from biochemical data, we used homology modeling to obtain a structural model of the SLI / SLV interaction. This model helps explain our kinetic results and allows us to propose novel interactions at the loop I / loop V interface.
RESULTS
Using kcat/KM values to investigate SLI recognition
To better understand the role of U700 and other SLV loop features in substrate recognition, we initiated kinetic studies of transcleavage with the VS ribozyme [7]. We used the previously-characterized AvaI VS ribozyme (Rz; [7]), either the wild-type RNA (Fig. 2.1A) or RNAs containing a variety of mutations in the SLV loop (Fig. 2.3). Binding of this ribozyme to the wild-type SLI substrate involves the loop I / loop V interaction and a conformational change from the unshifted state to the shifted state, which activates the substrate for catalysis (Fig. 2.1B). The SLI(T) substrate (S) was selected for kinetic studies, because it can only adopt a preshifted active state (Fig. 2.1C; [21, 23]).
The second-order rate constant (kcat/KM) for the reaction of Rz with S was
determined in single-turnover experiments [24], as illustrated for the U700G mutant Rz in Fig. 2.4. Cleavage experiments were performed with 32P-labeled S and excess Rz, and the conversion of substrate into product was monitored by gel electrophoresis (Fig. 2.4A). For these experiments, the natural log of the fraction of remaining substrate was plotted against the time of the reaction, and the value of the first-order rate constant, kobs,
was derived from the slope of the graph, as shown in Fig. 2.4B. For the wild-type and mutant Rz, a linear relationship was observed when kobs was plotted as a function of Rz
concentration, and the values of catalytic efficiency (kcat/KM) were obtained from the
slope of that graph (Fig. 2.4C). Table 2.1 provides a summary of the kcat/KM values and
of the relative catalytic efficiencies of the wild-type (WT) and mutant (MUT) ribozymes [(kcat/KM)WT/(kcat/KM)MUT].
The parameter kcat/KM is an important kinetic constant that combines both the
rate and the binding terms, and values of kcat/KM of wild-type and mutant enzymes can
be compared to address the role of specific residues in catalysis. For enzymes mutated at residues involved only in substrate binding and not in the reaction chemistry, kcat/KM
values of wild-type and mutant enzymes have been used to analyze the contribution of the mutated residues to the stabilization of the enzyme-substrate complex [25, 26]. Biochemical studies have implicated residues from the SLV loop in SLI substrate recognition [20], and there is no evidence that SLV residues participate in the reaction chemistry [19]. Therefore, kcat/KM values measured for VS ribozymes carrying
mutations in the SLV loop can be used to analyze the contribution of SLV loop residues to SLI recognition. SLI recognition is defined here as the binding of the SLI substrate to the trans-cleaving VS ribozyme and includes all ribozyme-substrate interactions.
Substrate recognition of the preshifted SLI(T) substrate used for our studies should involve formation of the loop I / loop V interaction [20] and docking of SLI at the active site [10, 27]. Current data are consistent with the role of the SLV loop being limited to the loop I / loop V interaction [20], although it is possible that mutations of the SLV loop also affect the proper docking of SLI at the active site, either because this docking depends on SLV interaction(s) with the rest of the ribozyme or as a consequence of a perturbed loop I / loop V interface. In the context where an SLV mutation would disrupt proper SLI docking at the active site, both SLI binding and the rate of chemistry could be affected, and a quantitative analysis of the effect of the mutation on SLI binding would not be possible [28]. In addition, mutations in SLV could also affect the folding of the ribozyme and/or change the energy landscape of the reaction. Thus, the kcat/KM values obtained here can only be interpreted in a qualitative
manner; they provide information on the loop I / loop V interaction, but could also reflect other aspects of the ribozyme cleavage reaction.
Kinetic properties of VS ribozymes carrying mutations in the SLV loop
The values of kcat/KM indicate that mutation of U700 by A, C, or G does not
significantly disrupt ribozyme activity, since the ratios of (kcat/KM)WT/(kcat/KM)MUT are
less than 4 (Table 2.1). The U700G mutation is the least disruptive, followed by U700A, and U700C. Deletion of U700, however, severely affects ribozyme activity, since the kcat/KM of the ΔU700 mutant is reduced 140 fold compare to the wild-type value. The
kcat/KM value of ΔU700 is similar to that of the positive control C699G
[(kcat/KM)WT/(kcat/KM)MUT = 270], a mutant which is expected to disrupt the formation of
the loop I / loop V interaction [20].
Mutation of the loop-closing U695-A701 base pair by a C-G or a G-A base pair also does not significantly affect ribozyme activity, since (kcat/KM)WT/(kcat/KM)MUT
values of 4.1 and 2.7 are observed for the U695C/A701G and U695G mutants, respectively. However, deletion of U700 in the context of a loop-closing G-A base pair severely reduces ribozyme activity since a (kcat/KM)WT/(kcat/KM)MUT value of 74 was
observed for the U695G/ΔU700 mutant, a value similar to that of the ΔU700 mutant (Table 2.1), which contains the wild-type loop-closing U-A base pair.
Insertion of a single U residue between C699 and U700 does not significantly affect ribozyme activity, since a (kcat/KM)WT/(kcat/KM)MUT value of 3.9 was observed for
the +U^U700 mutant. However, the insertion of the UC dinucleotide between C699 and U700 severely reduces ribozyme activity as judged from the (kcat/KM)WT/(kcat/KM)MUT
value of 130 observed for the +UC^U700 mutant.
NMR studies of mutant SLV fragments
To help interpret the kinetic data of the mutant ribozymes, we characterized SLV fragments containing the equivalent mutations (Fig. 2.3) by NMR spectroscopy. In order to ascertain that SLV fragments formed a hairpin as in the folded ribozyme and not a duplex conformation (Fig. 2.5A), they were analyzed by 1D imino 1H NMR spectroscopy (not shown) and native gel electrophoresis (Fig. 2.5B). The NMR data indicated that, except for U700A, wild-type SLV and all mutants adopted a single conformation in the absence of Mg2+. In the presence of Mg2+, the NMR data indicated
the presence of a second conformation (>10%) for select mutants. As illustrated in Fig. 2.5B, the native gel data could be used to confirm that the hairpin conformation is favored for the majority of SLV mutants, but that a few mutants formed a mixed population of hairpin and duplex conformations. The presence of a significant
population of duplex prevented further structural characterization of these mutants (U700A, U700G, and +UC^U700) by NMR. For the U700A and the U700G mutants, there is a clear possibility for base pairing involving residue 700 (A or G) and U696 (Fig. 2.5) that stabilizes the duplex conformation of the SLV fragment. It is important to note that the kcat/KM values of the ribozyme mutants U700A and U700G are not
significantly lower than that of the wild-type ribozyme (Table 2.1). Therefore the observation that certain mutations produce duplexes with SLV fragments does not suggest that these same mutations produce misfolded and functionally impaired ribozymes. On the contrary, since all mutations were localized to the loop of SLV and hydroxyl radicals footprinting data indicate that SLV does not interact with other parts of the ribozyme in the absence of SLI [27], it is likely that structural changes caused by our mutations would only affect the loop of SLV and not the general folding of the ribozyme. To obtain local structural information on the mutants, we compared their NMR data with that of the wild-type SLV.
Previous NMR studies of an SLV RNA fragment indicated that the SLV loop adopts a loose non-canonical U-turn fold in the absence of Mg2+ (SLVfree), which becomes a compact canonical U-turn fold in the presence of Mg2+ (SLVMg; [15, 16]). In agreement with previous NMR studies of U-turn structures [29-31], unusually large chemical shift changes were associated with the formation of a canonical U-turn structure in SLVMg for two specific NMR signals, the 31P resonance of U696 3'-
phosphate (3'-P) and the 15N resonance of A698 N7 [15, 16]. The detection of unusual
31P and 15N chemical shifts in SLV RNA fragments was therefore used to identify the
formation of a canonical U-turn fold for the mutants being functionally characterized in this study (Fig. 2.3).
NMR structural probing of the wild-type and mutant SLV RNAs involved the collection and analysis of 1D 1H-decoupled 31P spectra and 2D 1H-15N long-range
HMQC spectra [32]. In the 1D 31P spectra of the wild-type SLV RNA, distinct downfield-shifted signals were observed in the presence but not the absence of Mg2+ (Fig. 2.6A). Based on previous assignment, the 31P signal of U696 3'-P is the most
downfield-shifted signal, ~1.5 ppm downfield from the main cluster of 31P signals [16].
The unusual chemical shift of U696 3'-P was previously associated with formation of a canonical U-turn structure for SLVMg. In this structure, the 3'-P of U696 acts as the turning phosphate of the U-turn fold and is part of a negatively-charged pocket that